Cationic lipid-mediated enhancement of nucleic acid immunization of cats

ABSTRACT

The present invention relates to a method to introduce a nucleic acid molecule into a felid by administration of a nucleic acid-cationic lipid complex composition. The method includes the step of administering to the felid, by a parenteral route, a nucleic acid-cationic lipid complex to elicit and/or enhance an immune response. In one embodiment, this method enhances the immune response in a felid compared to a method in which a naked DNA vaccine is administered to a felid. Also provided is a method to deliver a nucleic acid to a felid. This method comprises parenterally administering to the felid a composition that includes a nucleic acid molecule complexed with a cationic lipid.

FIELD OF THE INVENTION

The present invention relates to a method to introduce a nucleic acidmolecule into a felid by administration of a nucleic acidmolecule-cationic lipid complex composition. In particular, the presentinvention relates to the parenteral administration of a nucleic acidmolecule-cationic lipid complex to elicit and/or enhance an immuneresponse to the protein encoded by the administered nucleic acidmolecule.

BACKGROUND OF THE INVENTION

Introduction of DNA into an animal for the purpose of eliciting animmune response is often referred to as DNA vaccination. DNA vaccinationrepresents a means of expressing an antigen in vivo for the generationof humoral and cellular immune responses. DNA vaccines employ genesencoding antigens, rather than using the proteins themselves, to induceimmune responses. The DNA, upon administration to the host, istranscribed and translated in vivo to produce an antigen. Processing andpresentation of the antigen stimulates the animal's immune system toelicit a humoral and/or cellular response to the antigen. This immuneresponse can potentially confer protective immunity to the animal.

DNA vaccines appear to have advantages over protein antigen-basedvaccines, standard “killed” pathogen vaccines, live, attenuatedvaccines, and recombinant viral vector vaccines. For example, DNAvaccines appear to be more effective in producing an antigen with aproperly folded, native three-dimensional conformation and in generatinga cellular immune response than are protein antigens. DNA vaccines alsodo not exhibit at least some of the safety problems of killed, live orvirally-vectored vaccines. For example, a killed virus preparation maycontain residual live viruses or may need to be mixed with reactogenicadjuvants, such as those associated with vaccine-related fibrosarcomasin cats, in order to stimulate an effective immune response. Anattenuated virus may mutate and revert to a pathogenic phenotype. Viralvector vaccines genetically engineered to express a gene encoding thedesired antigen may stimulate the production of antibodies that reactwith the virus as well; such antibodies may render futile any furtherattempt to use that virus as a vector, even with a different geneinsert. In contrast, DNA vaccines apparently are non-reactogenic and, ifthey elicit an immune response, that response is targeted against theantigen of choice.

DNA vaccines typically include a bacterial plasmid, a strong viralpromoter, the gene of interest, and a polyadenylation/transcriptionaltermination sequence. The plasmid is typically grown in bacteria,purified, dissolved in a saline solution, and then simply injected intoan animal. Current understanding of how to use DNA vaccines to generatean effective immune response, however, is not complete. Most of ourunderstanding of the mechanisms of DNA vaccine action is derived fromrodent studies. In mice, bone marrow-derived antigen-presenting cellshave been shown to induce cytotoxic T lymphocyte responses followingintramuscular inoculation of naked plasmid DNA. In some cases, DNAvaccination has also been shown to stimulate antigen-specificantibodies, some of which may be neutralizing antibodies. DNA vaccineshave also been administered to large animals, albeit with varyingdegrees of success. While there are some clear examples of DNA vaccineefficacy in large animals, other studies cite relatively weak responses,requirement for large amounts of DNA, or the need for multipleimmunizations. As such, it is apparent that further technologydevelopment is required to maximize DNA vaccine efficacy in humans andlarge animals.

Immune responses to DNA vaccination appear to vary according to thevehicle used with the DNA vaccine, the antigen expressed by the DNAvaccine, the route of administration, and the species of mammal intowhich the DNA vaccine is injected. Investigators have used differentvehicles and/or genes encoding cytokines and other stimulatory moleculesin an attempt to enhance the immune response to the antigens encoded byDNA vaccines with mixed success. Although cationic lipids have been usedto deliver nucleic acids to cells in vitro and in vivo, there is noconsensus in the literature about whether cationic lipids reproduciblyenhance the immunogenicity of DNA vaccines. Gregoriadis et al., 1997,FEBS Letters 402, 107, reported that intramuscular (I.M.) injection ofDNA encoding HBsA “entrapped” in cationic liposomes into mice elicitedan enhanced immune response compared to I.M. injection of “naked” DNAencoding HBsA, whereas DNA encoding HBsA merely “complexed” withcationic lipid generated a reduced immune response compared to “naked”DNA. Ishii et al., 1997, AIDS Research and Human Retroviruses 13,1421-1424, demonstrated enhanced immune responses to V3 peptidefollowing I.M., intraperitoneal (I.P.), intradermal (I.D.), intranasal(I.N.) or subcutaneous (S.Q.) administration to mice.

Other investigators, in contrast, found no enhancement of immuneresponses when cationic lipids were used as a vehicle for DNA vaccinesin mice. For example, Davis, et al., 1997, Vaccine 15, 849, found thatDNA vaccines encoding the Hepatitis B surface antigen formulated withvarying amounts of cationic lipids performed no better than DNA alone ininducing a humoral response in mice. Gramzinski, et al., 1998, MolecularMedicine 4, 109, reported that Aotus monkeys administered DNA vaccinesencoding HBsA either with or without cationic lipids (CELLFECTIN®, 10:1DNA:lipid) by I.M. injection did not seroconvert. Clearly, there is noconsensus regarding whether cationic lipids reproducibly act to elicitor enhance immune responses to DNA vaccines.

There also appears to be a high degree of variability of the efficacy ofDNA vaccines between different routes of administration. Ishii et al,ibid., for example, found in mice that I.M. and I.N. administration ofDNA vaccines generated approximately equivalent immune responses, butthat I.P. administration was less effective, and that I.D. and S.Q.administration routes were even less effective. Ishii et al, ibid.,found these differences to be consistent regardless of whether DNA wasused alone or formulated with cationic lipids. Yokoyama et al, 1996,FEMS Immuno Med Microbio 14, 221-230, showed that I.V. administration ofa DNA vaccine generated a better immune response than I.M.administration of the same vaccine in mice.

Taken together, these data indicate that there is a high degree ofvariability in the effectiveness of DNA vaccines and in the ability ofcationic lipids to enhance the effectiveness of DNA vaccines both withinand between species and routes of administration.

There are a number of diseases in cats which lead to significantmorbidity and mortality. It would be desirable to provide novel and safevaccines that would confer protective immunity to these diseases. Thatthere is still a need for such vaccines is underscored not only by theassociation of some feline vaccines with the development offibrosarcomas but also by the finding that I.M. administration of nakedDNA encoding either human growth hormone (hGH) or rabies virusglycoprotein G into domestic cats resulted in incomplete seroconversion,even after two immunizations (Osorio et al, 1999, Vaccine, in press).These results indicate that parenteral naked DNA vaccination efficacy incats is inferior to results obtained in mice, and that the efficacyachieved using naked DNA in cats is not sufficient to protect cats fromdisease. Thus, there remains a need to provide a method to elicit and toenhance the immune response to antigen encoded by DNA vaccines in cats.

SUMMARY OF THE INVENTION

The present invention relates to a method to elicit an immune responseto an antigen in a felid. This method includes the step of parenterallyadministering to the felid a composition comprising a nucleic acidmolecule encoding the antigen in which the nucleic acid molecule iscomplexed with a cationic lipid. In one embodiment, this method enhancesthe immune response in a fetid compared to a method in which a naked DNAvaccine is administered to a fetid. Also provided is a method to delivera nucleic acid molecule to a felid. This method comprises parenterallyadministering to the fetid a composition that includes a nucleic acidmolecule complexed with a cationic lipid.

DETAILED DESCRIPTION

The present invention relates to a method to elicit an immune responseto an antigen in a fetid. The method includes the step of parenterallyadministering to the fetid a composition comprising a nucleic acidmolecule encoding the antigen in which the nucleic acid molecule iscomplexed with a cationic lipid. The ability of such a method to elicitan immune response to the antigen encoded by the nucleic acid moleculeis new and surprising. Until recently, the general perception of thoseskilled in the art was that cationic lipids did not enhance the abilityof a nucleic acid molecule to elicit an immune response, compared to,for example, delivery of a naked, or unformulated, nucleic acid molecule(i.e., a nucleic acid molecule that is not complexed with, for example,a lipid or other transfection-facilitating agents). Recent studies,cited above, have provided conflicting results: although two studies inmice demonstrated that cationic lipids enhanced the ability of DNA toelicit an immune response, a third study concluded that cationiclipid-complexed DNA was no better than naked DNA at eliciting an immuneresponse. In addition, monkeys administered a nucleic acidmolecule-cationic lipid complex did not exhibit seroconversion to theantigen encoded by the nucleic acid molecule. Furthermore, the inventorshave demonstrated that while parenteral administration to a felid of anucleic acid molecule complexed with a cationic lipid results in thefelid successfully seroconverting in response to the antigen encoded bythe nucleic acid molecule, intranasal administration of such acomposition did not result in seroconversion. Thus, the ability todemonstrate seroconversion in cats parenterally administered a nucleicacid molecule complexed with a cationic lipid is completelyunpredictable based on previous studies and, as such, is inventive.

One embodiment of the present invention is the use of a compositioncomprising a nucleic acid molecule encoding an antigen complexed with acationic lipid to elicit an immune response in a felid. It is to benoted that the term “a” or “an” entity refers to one or more of thatentity; for example, a nucleic acid molecule, an antigen, and a cationiclipid refers to one or more nucleic acid molecules, antigens, andcationic lipids, respectively; or to at least one nucleic acid molecule,antigen, and cationic lipid, respectively. As such, the terms “a” (or“an”), “one or more” and “at least one” can be used interchangeablyherein. It is also to be noted that the terms “comprising”, “including”,and “having” can be used interchangeably. Furthermore, a member of agroup that is “selected from the group consisting of” refers to one ormore of members of that group, including combinations thereof.

A nucleic acid molecule of the present invention also referred to hereinas a nucleic acid, can be DNA or RNA. In one embodiment, a nucleic acidmolecule encodes an antigen that elicits an immune response in a felid.As such, a nucleic acid molecule can simply be a molecule that encodessuch an antigen, i.e., a coding region, or the nucleic acid molecule cancomprise a coding region operatively linked to a regulatory sequence. Asused herein, the phrase operatively linked refers to the joining of acoding region to one or more regulatory sequences such that the codingregion is expressed using such regulatory sequence(s) in a felid.Examples of such regulatory sequences include transcription controlsequences and translation control sequences that can be recognized byfelid cellular mechanisms in order to effect transcription andtranslation of a coding region. Transcription control sequences aresequences that control the initiation, elongation, and termination oftranscription (e.g., promoters, enhancers, introns, polyA sites,terminators). Translation control sequences control the initiation,elongation and termination of translation. Additional regulatorysequences include signal sequences that effect secretion of a proteinfrom a cell and a combination of a signal sequence and a transmembranesequence (i.e., membrane anchoring domain) that causes a protein to bepartially extracellular and partially retained in the membrane and/orcytoplasm. A preferred nucleic acid molecule of the present invention isa plasmid or viral genome that includes a coding region for the desiredantigen operatively linked to strong eukaryotic regulatory sequences,including a strong promoter and strong transcriptiontermination/polyadenylation sequences. A preferred plasmid can replicatein bacteria. Procedures by which such a nucleic acid molecule isproduced are known to those skilled in the art, and are disclosed, forexample, in Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Labs Press. Appropriate plasmids are known inthe art, and may include, but are not limited to, pUC 19 andBLUESCRIPT®. A preferred plasmid is pUC19. Appropriate regulatorysequences are known to those skilled in the art. For example, a suitablepromoter includes, but is not limited to the cytomegalovirus immediateearly promoter (CMV IE) with or without intron A, a long terminal repeat(LTR) promoter from a retrovirus, or a strong cellular promoter such asβ-actin, with CMV IE with intron A being preferred. Similarly, suitabletranscription termination sequences include, but are not limited to,bovine growth hormone, SV40 virus or rabbit beta-globin polyadenylationsequences, with a bovine growth hormone sequence being preferred.

A suitable antigen is any antigen that effects an immune response, andas such includes allergens and autoantigens as well as other antigens.An antigen, as used herein, can refer to the full-length antigen or anyportion thereof that is capable of eliciting an immune response.Preferred antigens are those that elicit an immune response thatprotects an animal from disease. Examples of such antigens include, butare not limited to, a protozoan parasite antigen, a helminth parasiteantigen, an ectoparasite antigen, a fungal antigen, a bacterial antigen,and a viral antigen. Examples of viral antigens include, but are notlimited to, antigens from adenoviruses, caliciviruses, coronaviruses,distemper viruses, hepatitis viruses, herpesviruses, immunodeficiencyviruses, infectious peritonitis viruses, leukemia viruses, oncogenicviruses, papilloma viruses, parainfluenza viruses, parvoviruses, rabiesviruses, and reoviruses, as well as other cancer-causing orcancer-related viruses. Examples of bacterial antigens include, but arenot limited to, antigens from Actinomyces, Bacillus, Bacteroides,Bordetella, Bartonella, Borrelia, Brucella, Campylobacter,Capnocytophaga, Clostridium, Corynebacterium, Coxiella, Dermatophilus,Enterococcus, Ehrlichia, Escherichia, Francisella, Fusobacterium,Haemobartonella, Helicobacter, Klebsiella, L-form bacteria, Leptospira,Listeria, Mycobacteria, Mycoplasma, Neorickettsia, Nocardia,Pasteurella, Peptococcus, Peptostreptococcus, Proteus, Pseudomonas,Rickettsia, Rochalimaea, Salmonella, Shigella, Staphylococcus,Streptococcus, and Yersinia. Examples of fungal antigens include, butare not limited to, antigens from Absidia, Acremonium, Alternaria,Aspergillus, Basidiobolus, Bipolaris, Blastomyces, Candida, Chlamydia,Coccidioides, Conidiobolus, Cryptococcus, Curvalaria, Epidermophyton,Exophiala, Geotrichum, Histoplasma, Madurella, Malassezia, Microsporum,Moniliella, Mortierella, Mucor, Paecilomyces, Penicillium, Phialemonium,Phialophora, Prototheca, Pseudallescheria, Pseudomicrodochium, Pythium,Rhinosporidium, Rhizopus, Scolecobasidium, Sporothrix, Stemphylium,Trichophyton, Trichosporon, and Xylohypha. Example of protozoan andhelminth parasite antigens include, but are not limited to, antigensfrom Babesia, Balantidium, Besnoitia, Cryptosporidium, Eimeria,Encephalitozoon, Entamoeba, Giardia, Hammondia, Hepatozoon, Isospora,Leishmania, Microsporidia, Neospora, Nosema, Pentatrichomonas,Plasmodium, Pneumocystis, Sarcocystis, Schistosoma, Theileria,Toxoplasma, and Trypanosoma, Acanthocheilonema, Aelurostrongylus,Ancylostoma, Angiostrongylus, Ascaris, Brugia, Bunostomum, Capillaria,Chabertia, Cooperia, Crenosoma, Dictyocaulus, Dioctophyme, Dipetalonema,Diphyllobothrium, Diplydium, Dirofilaria, Dracunculus, Enterobius,Filaroides, Haemonchus, Lagochilascaris, Loa, Mansonella, Muellerius,Nanophyetus, Necator, Nematodirus, Oesophagostomum, Onchocerca,Opisthorchis, Ostertagia, Parafilaria, Paragonimus, Parascaris,Physaloptera, Protostrongylus, Setaria, Spirocerca, Spirometra,Stephanofilaria, Strongyloides, Strongylus, Thelazia, Toxascaris,Toxocara, Trichinella, Trichostrongylus, Trichuris. Uncinaria, andWuchereria. Examples of ectoparasite antigens include, but are notlimited to, antigens (including protective antigens as well asallergens) from fleas; ticks, including hard ticks and soft ticks;flies, such as midges, mosquitos, sand flies, black flies, horse flies,horn flies, deer flies, tsetse flies, stable flies, myiasis-causingflies and biting gnats; ants; spiders, lice; mites; and true bugs, suchas bed bugs and kissing bugs. Additional examples of suitable allergensinclude food, grass, weed, tree pollen, other animal and other plantallergens.

Preferred antigens include, but are not limited to, a calicivirusantigen, a coronavirus antigen, a herpesvirus antigen, animmunodeficiency virus antigen, an infectious peritonitis virus antigen,a leukemia virus antigen, a panleukopenia virus antigen, a parvovirusantigen, a rabies virus antigen, a Bartonella antigen, a Yersiniaantigen, a Dirofilaria antigen, a Toxoplasma antigen, a tumor antigen, aflea antigen, a flea allergen, a midge antigen, a midge allergen, a miteantigen, a mite allergen, a ragweed allergen, a ryegrass allergen, a catallergen, a dog allergen, a Bermuda grass allergen, a Johnson grassallergen, or a Japanese cedar pollen allergen. Particularly preferredantigens include a rabies virus glycoprotein G antigen; heartworm PLA2,P39, P4, P22U, Gp29, astacin, cysteine protease, macrophage migrationinhibitory factor, venom allergen, TPX-1, TPX-2, transglutaminase,ankyrin, asparaginase, calreticulin, cuticulin, and aromatic amino aiddecarboxylase antigens; flea serine protease, cysteine protease,aminopeptidase, serpin, carboxylesterase, juvenile hormone esterase,chitinase, epoxide hydrolase, ecdysone, ecdysone receptor, andultraspiracle protein antigens; flea salivary antigens; Yersinia F1 andV antigens; and Toxoplasma gondii antigens such as those disclosed inPCT Patent Publication No. WO 99/32633, published Jul. 1, 1999, byMilhausen et al. Additional examples of suitable and preferred allergensare disclosed in U.S. Pat. No. 5,945,294, issued Aug. 31, 1999, by Franket al. (U.S. Pat. No. 5,945,294); U.S. Pat. No. 5,958,880, issued Sep.28, 1999, by Frank et al. (U.S. Pat. No. 5,958,880); PCT PatentPublication No. WO 98/45707, published Oct. 15, 1998, by Frank et al.(WO 98/45707); and PCT Patent Publication No. WO 99/38974, publishedAug. 5, 1999, by Weber et al. (WO 99/38974).

One embodiment of the present invention is a composition comprising anucleic acid molecule-cationic lipid complex that further comprises aheterologous nucleic acid molecule encoding an immunomodulator. Such animmunomodulator-encoding nucleic acid molecule can be contained withinthe same nucleic acid molecule encoding the antigen of the presentinvention, or can exist as a separate nucleic acid molecule, which canbe on the same or separate plasmid or viral genome. The presentinvention also includes Suitable immunomodulators include compounds thatenhance certain immune responses as well as compounds that suppresscertain immune responses. Compounds that enhance the immune responseinclude compounds that preferentially enhance humoral immunity as wellas compounds that preferentially enhance cell-mediated immunity.Suitable compounds can be selected depending on the desired outcome.Suitable immunomodulators include, but are not limited to, cytokines,chemokines, superantigens, co-stimulatory molecules, adhesion molecules,and other immunomodulators as well as compounds that induce theproduction of such immunomodulators. Examples of such compounds include,but are not limited to, granulocyte macrophage colony stimulating factor(GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophagecolony stimulating factor (M-CSF), colony stimulating factor (CSF),erythropoietin (EPO), interleukin 2 (IL-2), interleukin 3 (IL-3),interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6),interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10 (IL-10),interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 18 (IL-18),interferon gamma, interferon gamma inducing factor I (IGIF),transforming growth factor beta (TGF-β), RANTES (regulated uponactivation, normal T-cell expressed and presumably secreted), macrophageinflammatory proteins (e.g., MIP-1 alpha and MIP-1 beta), Leishmaniaelongation initiating factor (LEIF), B7-1, B7-2, CD40, CD40 ligand,ICAM-1, and VCAM.

A composition of the present invention includes a cationic lipidcomplexed with a nucleic acid molecule encoding an antigen in order toelicit or enhance an immune response to the antigen. As used herein, acationic lipid is a lipid which has a cationic, or positive, charge atphysiologic pH. Cationic lipids can have a variety of forms, includingliposomes or micelles. Whether a cationic lipid occurs primarily as aliposome or a micelle can be manipulated by methods known in the art;for example, a freezing and thawing of cationic lipids in aqueoussolution will encourage formation of liposomes, rather than micelles. Anucleic acid molecule complexed with a cationic lipid may also bereferred to as a nucleic acid molecule-cationic lipid complex, alipoplex or a complex of the present invention. A complex of the presentinvention that elicits an immune response is a complex of a nucleic acidmolecule which encodes an antigen with a cationic lipid. As used herein,the term complexed with, which is equivalent to complexed to, refers toany method by which a nucleic acid molecule interacts (e.g. binds, comesinto contact with a cationic lipid.) Such an interaction can include,but is not limited to encapsulation of a nucleic acid molecule into acationic liposome, association of a nucleic acid molecule and cationiclipid characterized by non-covalent, ionic charge interactions, andother types of associations between nucleic acid molecules and cationiclipids known by those skilled in the art. It is preferred that cationiclipids have a cationic group, such as a quaternary amine group, and oneor more lipophilic groups, such as saturated or unsaturated alkyl groupshaving from about 6 to 30 carbon atoms. Cationic lipid compositionssuitable for use in the present invention include lipid compositionscomprised of one type of lipid, or lipid compositions comprised of morethan one type of lipid. If there is more than one type of lipid presentin a lipid composition, it is necessary that the overall net charge ofthe lipid composition is cationic, i.e. positive; however, as long asthe overall net charge of the lipid composition is cationic, individuallipid types may be neutral or even anionic in charge. A composition ofthe present invention includes a cationic lipid that is suitable inaccordance with the present invention. Cationic lipids suitable for usein the present invention include commercially available cationic lipids,for example DOTMA, available under the trademark name of LIPOFECTIN®,available from Life Technologies Inc., (LTI), Gaithersburg, MD and DDAB,available from Boehringer-Mannheim, Indianapolis, Ind. In addition,suitable cationic lipids can be synthesized as described in theliterature; see, for example, Felgner et al., 1987, PNAS 84 7413-7417regarding the preparation of DOTAP; Douar et al, 1996, Gene Ther 3(9),789-796 regarding the preparation of Lipid 67; Wheeler et al., 1996,Biochim Biophys Acta 1280(1), 1-11 regarding the preparation of DMRIE;McLean et al., 1997 Am J Physiol 273, H387-404 regarding the preparationof DOTIM; and Hofland et al., 1997, Pharm Res 14(6), 742-749 regardingthe preparation of DOSPA. Other suitable cationic lipid compounds aredescribed in the literature. See, for example, Stamatatos et al., 1988,Biochemistry 27, 3917-3925 and Eibl, et al., 1979, Biophysical Chemistry10, 261-271. Preferred cationic lipids include the class of lipids knownas tetramethyltetraalkyl spermine analogs, described by McCluskey etal., (1998), Antisense and Nucleic Acid Drug Development, vol. 8, pp401-414. Lipids of this type include tetramethyltetralaurylspermine,tetramethyltetramyristylspermine, tetramethyltetrapalmitoylspermine, andtetramethyltetraoleoylspermine. The following lipids, obtained from LTIare of the tetramethyltetraalkyl spermine class, with the alkyl groupscontaining fatty acid chains of length longer than oleic acid. Theselipids are denoted as LTI lipids 4251-781-1, 4251-106-3, 4518-52,D304-200, 4521-52-3, 4251-106-4, 4251-781-2, 4518-53, 4518-31,4519-30,4519-34, and 2518-111. Preferred cationic lipids include LTIlipid 4251-781-1, LTI lipid 4251-106-3, and LTI lipid 4518-52. In oneembodiment, tetramethyltetraalkyl spermine lipids are formulated with aneutral lipid, such as dioleylphosphatidyl-ethanolamine (DOPE).

A nucleic acid molecule-cationic lipid complex can be formed by usingtechniques known to those skilled in the art, examples of which aredescribed in the Examples section. A complex can be formed, for example,by adding a cationic lipid solution to a nucleic acid molecule,preferably an endotoxin-free nucleic acid molecule, at concentrationsappropriate for the present invention, and mixing, for example bypipetting. Preferable nucleic acid molecule-to-cationic lipid ratios arefrom about 10:1 weight nucleic acid molecule: weight cationic lipid,(e.g. microgram (μg) nucleic acid molecule to μg cationic lipid) toabout 1:10 weight nucleic acid molecule: weight cationic lipid. Morepreferable are ratios from about 1:2 weight of nucleic acid molecule:cationic lipid to about 4:1 weight of nucleic acid molecule: cationiclipid. In a preferred embodiment, the nucleic acid molecule-cationiclipid complex is incubated at room temperature for about 30 minutesbefore administration. A nucleic acid molecule-cationic lipid complexcan be dehydrated and rehydrated using techniques known to those skilledin the art; for example, the complex can be frozen in liquid nitrogenand lyophilized at 150 milliTorr, then reconstituted in solution forinjection.

A dose of a nucleic acid molecule-cationic lipid complex to administerto a cat can be reported as the amount of nucleic acid moleculeadministered to a cat. A preferred dose of a nucleic acidmolecule-cationic lipid complex to administer to a cat includes from atleast one nanogram (ng) of nucleic acid to about 10 milligram (mg) ofnucleic acid molecule. More preferred is a dose range that includes fromabout 1 μg nucleic acid molecule to about 1 mg of nucleic acid molecule.Particularly preferred is a dose ranging from about 75 μg of a nucleicacid molecule to about 300 μg of a nucleic acid molecule.

A nucleic acid molecule-cationic lipid complex composition of thepresent invention can be formulated in an excipient that the animal tobe treated can tolerate. As such, the present invention includesadministration of a composition comprising a nucleic acidmolecule-cationic lipid complex, wherein the composition furthercomprises an excipient. Examples of such excipients include water,saline, Ringer's solution, dextrose solution, Hank's solution, and otheraqueous physiologically balanced salt solutions. Other usefulformulations include suspensions containing viscosity enhancing agents,such as sodium carboxymethylcellulose, sorbitol, mannitol, or dextran.Excipients can also contain minor amounts of additives, such assubstances that enhance isotonicity and chemical stability. Examples ofbuffers include phosphate buffer, bicarbonate buffer and Tris buffer.Standard formulations can either be liquid injectables or solids whichcan be taken up in a suitable liquid as a suspension or solution forinjection. Thus, in a non-liquid formulation, the excipient can comprisedextrose, human serum albumin, preservatives, etc., to which sterilewater or saline can be added prior to administration.

In one embodiment of the present invention, the nucleic acidmolecule-cationic lipid complex can also include an adjuvant and/or acarrier. One advantage of a nucleic acid molecule-cationic lipid complexis that adjuvants and carriers are not required to produce a compositionthat administration thereof will elicit an immune response. However, itshould be noted that use of adjuvants or carriers is not precluded bythe present invention. Adjuvants are typically substances that generallyenhance the immune response of an animal to a specific antigen. Suitableadjuvants include, but are not limited to, other bacterial cell wallcomponents; aluminum-based salts; calcium-based salts; silica;polynucleotides; toxins, such as cholera toxin; toxoids, such as choleratoxoid; serum proteins; other viral coat proteins; otherbacterial-derived preparations; block copolymer adjuvants, such asHunter's Titermax™ adjuvant (Vaxcel™, Inc. Norcross, Ga.); Ribiadjuvants (available from Ribi ImmunoChem Research, Inc., Hamilton,Mont.); and saponins and their derivatives, such as Quil A (availablefrom Superfos Biosector A/S, Denmark). Carriers are typically compoundsthat increase the half-life of a therapeutic composition in the treatedanimal. Suitable carriers include, but are not limited to, polymericcontrolled release formulations, biodegradable implants, liposomes,bacteria, viruses, oils, esters, and glycols.

An immune response to an antigen includes a humoral, i.e. antibody,response to that antigen and/or a cell-mediated response to thatantigen. Methods to measure an immune response are known to thoseskilled in the art; examples of such methods are disclosed herein. Ifone or both types of immune response are present, they may protect thefelid from disease caused, for example, by the agent from which theantigen was derived. In accordance with the present invention, theability of an antigen derived from a disease-causing agent to protect ananimal from a disease caused by that disease-causing agent or across-reactive agent refers to the ability of a nucleic acidmolecule-cationic lipid complex of the present invention to treat,ameliorate and/or prevent disease caused by the disease-causing agent orcross-reactive agent, preferably by eliciting an immune response againstthe antigen derived from the disease-causing agent. It is to be notedthat an animal may be protected by a composition of the presentinvention even without the detection of a humoral or cell-mediatedresponse to the antigen. Protection can be measured by methods known tothose skilled in the art, such as by challenging an animal with theagent against which the animal has mounted a putative immune response.In certain cases, the antibody titer of an animal can be used todemonstrate protection. For example, it is known that animals thatelicit an antibody response against a rabies glycoprotein G antigen areprotected if their sera exhibits a rapid focus fluorescent inhibitiontest (RFFIT) titer of rabies virus neutralizing antibodies of greaterthan 1:5. As used herein, an animal that elicits an immune response toan antigen is an animal that has been immunized with that antigen.

The biological mechanism for eliciting and/or enhancing an immuneresponse by the use of a nucleic acid molecule-cationic lipid complexcomposition of the present invention has not been elucidated, but,without being bound by theory, the inventors believe that the mechanismis likely related to the ability of these compositions to protect DNAfrom nuclease attack, to facilitate the transfection of both musclecells and professional antigen presenting cells (APC) in vivo, toincrease levels of expression in transfected cells, and/or to distributeDNA to lymphoid organs.

A felid, as used herein, is a member of the family Felidae. Examples offelids include domestic cats, wild cats, and zoo cats. Examples of cats,include, but are not limited to, domestic cats, lions, tigers, leopards,panthers, cougars, bobcats, lynx, jaguars, cheetahs, and servals. Apreferred cat to immunize is a domestic cat. The term cat(s) andfelid(s) are used interchangeably herein.

As used herein, parenteral administration means administration notthrough the alimentary canal (e.g. oral administration), but rather byinjection through some other route, including but not limited to routessuch as subcutaneous, intramuscular (I.M.), intravenous (I.V.),intraperitoneal (I.P.), intradermal (I.D.), intraorbital, intracapsular,intraspinal, and intrasternal. Parenteral administration includes, butis not limited to, administration by any route that includes use of aneedle to insert material into the body. Parenteral administration alsoincludes uses of devices other than a syringe and needle to insertmaterial through the skin and or mucosal surfaces into the body,including but not limited to the BIOJECTOR®, POWDERJECT, and MEDIJECT®needleless injection systems. A preferred route of administrationincludes intramuscular administration using a needle and syringe.

Acceptable protocols to administer therapeutic compositions in aneffective manner include individual dose size, number of doses, andfrequency of dose administration. Typically, the first administration ofa composition intended to elicit an immune response is called theprimary (or prime) administration, also known as the pre-boost.Additional administrations intended to “boost” or increase an immuneresponse to an antigen are termed booster administrations. Determinationof a protocol to elicit an immune response in a cat using a nucleic acidmolecule-cationic lipid complex of the present invention can beaccomplished by those skilled in the art. In one embodiment of thepresent invention, a nucleic acid molecule encoding a desired antigencomplexed with cationic lipid need only be administered once by a routeappropriate to the present invention (e.g. parenteral) to stimulate animmune response against the antigen. In a preferred embodiment, such anadministration protects the felid from the agent from which the antigenwas derived or from an agent against which the immune response iscross-protective.

In one embodiment, administration of a complex of the present inventionto a felid in order to elicit an immune response actually enhances theimmune response generated by the felid as compared to the immuneresponse generated upon administration of a naked DNA vaccine to afelid, wherein the naked DNA vaccine consists essentially of a naked DNAmolecule; i.e., a DNA molecule that is not complexed with lipids.Finding that a complex of the present invention enhances an immuneresponse is surprising both in view of the conflicting studies known tothose skilled in the art as described herein as well as in view of thestudies described in more detail in the Examples, in whichadministration of naked DNA vaccines to cats elicited immune responsesin only some cats within each group, or population, tested, whereasadministration of a complex of the present invention could result in upto 100% seroconversion of all cats in a population tested. As usedherein, enhancement of the immune response can include increasing theamount, or titer, of antibody elicited by a complex of the presentinvention that encodes an antigen to the desired antigen and/or agentfrom which the antigen was derived as compared to the titer of antibodygenerated by a naked DNA vaccine that encodes the same antigen. In oneembodiment, such an enhancement can be induction of no antibody titerwith a naked DNA vaccine to induction of a protective antibody titerwith a complex of the present invention. Enhancement of an immuneresponse can also refer to augmentation of the cell-mediated response tothe antigen and/or agent encoded by a complex of the present inventionas compared to the response generated by a naked DNA vaccine encodingthe same antigen. Enhancement of immune response can also includeconferring or augmenting protection from disease by a complex of thepresent invention compared to the protection, if any, conferred by anaked DNA vaccine encoding the same antigen. In one embodiment,enhancement of the immune response includes increasing the likelihood ofa cat seroconverting in response to antigen encoded by a complex of thepresent invention in comparison to the likelihood of the cat respondingto the same antigen encoded by a naked DNA vaccine. In other words, in agroup of cats being vaccinated with a complex of the present invention,a greater number of cats will seroconvert in response to antigen encodedby the complex rather than to the same antigen encoded by a naked DNAvaccine. Preferably, the likelihood that a cat will seroconvert whenadministered a single dose of a complex of the present invention thatencodes an antigen is at least about 50%, preferably at least about 75%,more preferably at least about 90% and even more preferably at leastabout 100%. In the case where a primary and booster administration ofthe complex is administered, the likelihood that a cat will seroconvertis preferably at least about 75%, more preferably at least about 90%,and even more preferably at least about 100%.

The present invention includes a method to administer a nucleic acidmolecule to a felid. The method includes the step of parenterallyadministering a composition comprising said nucleic acid moleculecomplexed with a cationic lipid. Such a nucleic acid molecule can encodeeither a protein or a RNA molecule. In one embodiment, the nucleic acidmolecule encodes a protein or RNA molecule that, when expressed at anappropriate level, has a protective effect upon the cat. As used herein,a protein refers to a full-length protein or any portion thereof that isat least about 5 amino acids in length and has a useful function,including, but not limited to, ability to elicit an immune response,elicit an immunomodulatory effect (e.g., an immunomodulator thatstimulates or reduces the immune response), effect gene therapy, effectenzyme activity, or otherwise effect cell division, differentiation,development and cell death. As used herein, a RNA molecule refers to anyRNA species that can be encoded by a nucleic acid molecule, including,but not limited to antisense RNA, a molecule capable of triple helixformation, a ribozyme, or other nucleic acid-based drug compound. Assuch, any protein or RNA molecule that can be expressed at anappropriate level in a cat, which protects a cat from disease, would beincluded in this invention. Diseases from which to protect a felidinclude, but are not limited to, infectious diseases, genetic diseases,oncological diseases, and other metabolic diseases, including diseasesthat lead to abnormal cell growth, degenerative processes, andimmunological defects. Compositions of the present invention can protectanimals from a variety of diseases including, but not limited to,allergies, arthritic diseases, autoimmune diseases, cancers,cardiovascular diseases, graft rejection, hematopoietic disorders,immunodeficiency diseases, immunoproliferative diseases,immunosuppressive disorders, infectious diseases, inflammatory diseases,jaundice, septic shock, and other immunological defects, as well asother genetic or metabolic defects. Methods to produce and use acomposition comprising any nucleic acid molecule of the presentinvention complexed with any cationic lipid of the present invention areas described herein.

The following examples are provided for the purposes of illustration andare not intended to limit the scope of the present invention.

EXAMPLES Example 1

This Example demonstrates the production of a nucleic acid molecule ofthe present invention.

A nucleic acid molecule encoding human growth hormone (hGH) wasconstructed using plasmid pHGH107 (available from American Type CultureCollection, Manassis, Va.), which encodes hGH amino-acids 1-191, as apolymerase chain reaction (PCR) template. The hGH open reading frame wasamplified by PCR using Pfu DNA polymerase (available from Stratagene, LaJolla, Calif.) and the following forward and reverse primers: 5′TTCCCAACTATACCACTATCTCGTCTA 3′ (SEQ ID NO:1) and 5′CTAGAAGCCACAGCTGCCCTCCACAGAG 3′ (SEQ ID NO:2). The PCR productcontaining the sequence encoding the mature hGH product was ligated intothe NaeI site of a plasmid containing the human cytomegalovirusimmediate early promoter, a translation control sequence, a sequenceencoding the signal peptide coding sequence from human tissueplasminogen activator, and a bovine growth hormone poly A sequence. Theexpression of hGH from this plasmid was confirmed following transfectionof cells in vitro and was detected using a chemiluminescence assay kit(available from Nichols Institute Diagnostics, San Juan Capistrano,Calif.).

A nucleic acid molecule encoding the rabies virus glycoprotein G wasdescribed previously and contains the CMV intron A promoter, the rabiesglycoprotein G coding sequence, and the bovine growth hormonepolyadenylation sequence. See Osorio, et al. (1999) Vaccine, in press.

Example 2

This Example describes the production of a nucleic acidmolecule-cationic lipid complex of the present invention.

Endotoxin-free nucleic acid molecules encoding hGH or rabiesglycoprotein G were prepared using a commercial kit (Qiagen, Inc.,Valencia, Calif.) and the resulting nucleic acid molecules weredissolved in endotoxin-free 10 mM Tris-HCl, pH 7.5, 1 mM EDTA at 2 mgper milliliter (ml) to form a hGH nucleic acid molecule solution and arabies gG nucleic acid molecule solution, respectively. Cationic lipids4251-106-3 (also known as 106-3), 4251-781-1 (also known as 781-1), and4518-52 were obtained from Life Technologies, Inc. (LTI), Gaithersburg,Md. A nucleic acid molecule-cationic lipid complex was formed by adding250 μl of the respective cationic lipid solution to 250 μl of therespective nucleic acid molecule solution, followed by immediate mixingby pipetting. The concentrations of the cationic lipid solutions and ofthe nucleic acid molecule solutions used were adjusted to give thedesired amounts and ratios of nucleic acid molecules to cationic lipidsdescribed elsewhere in the Examples. The mixture was incubated at roomtemperature for 30 minutes before administration. For dehydration andrehydration of a nucleic acid molecule-cationic lipid complex, thecomplex was frozen in liquid nitrogen and lyophilized at 150 mTorr, thenreconstituted in the original volume of sterile water for injection.

Example 3

This Example describes a method for administering a nucleic acidmolecule-cationic lipid complex of the present invention to a fetid.

Primary and booster administrations of nucleic acid molecule-cationiclipid complexes prepared as described in Example 2 were injectedintramuscularly into the semitendinosus or semimembranosus muscle ofdomestic cats. Each dose was divided into two equal portions andadministered bilaterally into each leg. Sera samples were collectedevery 10 days for antibody determination.

Example 4

This Example describes methods to measure immune responses generated inresponse to the administration of nucleic acid molecule-cationic lipidcomplexes of the present invention.

Antibody responses specific for hGH were determined by ELISA. Briefly,ELISA plate wells were coated with 0.4 micrograms (μg) hGH protein perwell (hGH protein available from Genzyme Diagnostics, San Carlos,Calif.) and incubated overnight at 4° C. Unbound antigen was aspiratedand the plate was blocked with 2% skimmed milk for 1 hour at 37° C.ELISA plates were washed 3 times with TBS-Tween (150 milliMolar (mM)NaCl, 50 mM Tris-HCl (pH 8.0), 0.1% TWEEN-20) and serially diluted serasamples from vaccinated cats were added and incubated at 37° C. for 1hour. Plates were washed 3 times with TBS-Tween. A biotin conjugatedmonoclonal anti-cat IgG (1:30,000) (available from Sigma-Aldrich, St.Louis, Mo.), was added and incubated for 1 hour at 37° C., followed bythe addition of EXTRAVIDIN®-horseradish perokidase diluted 1:1000,available from Sigma-Aldrich, St. Louis, Mo. After a final incubation at37° C., for 1 hour, the plates were washed and an o-phenylenediaminedihydrochloride substrate solution, available from Sigma-Aldrich, wasadded and the plates incubated at room temperature for 30 minutes forcolor development. The plates were read at 450 nm.

Rabies virus-specific neutralizing antibody response were determinedusing the Rapid Fluorescent Focus Inhibition test (RFFIT) at theDepartment of Veterinary Diagnostics, Kansas State University.

T cell proliferation assays were carried out in the following manner.Heparinized blood samples were collected from cats a week afteradministration of a booster injection as described in Example 5. Thelymphocytes were isolated from the blood samples using a percollgradient (Sigma Chemicals, St Louis, Mo.). The isolated lymphocytes wereresuspended in RPMI 1640 (Sigma Chemical) containing 5% normal catserum, 2 mM L-glutamine (Life Technologies, Bethesda, Md.), 1 mM sodiumpyruvate (Life Technologies), 50 μM 2-mercaptoethanol (LifeTechnologies), 5 μg/mL gentamycin (Sigma Chemical), 0.1 mM MEMnon-essential amino acids (Life Technologies), and 1% essential aminoacids (Life Technologies) plated at a density of 2×10⁵ cells/well andtreated with various concentrations of recombinant human growth hormone(hGH) (Genzyme Diagnostics, Boston, Mass.) for a total of 3 or 5 days.Each group of cell samples contained a negative control (media alone)and a positive control (Concanavalin A, Sigma Chemicals). Cells werepulsed at time of measurement with 0.5 μCurie of tritiated thymidine(ICN Pharmaceuticals) per well. T cells that were specific for hGHproliferated in response to added hGH and incorporated the tritiatedthyrnidine into their DNA. The amount of incorporated tritium wascounted 16 to 18 hours post-pulse in a scintillation counter. Data wasreported as the stimulation index, which was derived by dividing thecounts per minute obtained from the samples divided by the counts perminute obtained from the negative control.

Example 5

This Example compares the immune response elicited using a nucleic acidmolecule encoding hGH complexed with either LTI lipid 781-1 or LTI lipid106-3 to the immune response elicited using a naked DNA vaccine encodinghGH in cats.

The hGH nucleic acid molecule was complexed with LTI lipid 781-1 at alipid-to-DNA ratio (μ:μ) of 0.5:1.0, and formulated with LTI lipid 106-3at a lipid-to-DNA ratio of 1:1, as described in Example 2. The naked DNAvaccine consisted of the hGH nucleic acid molecule prepared as describedin Example 2 dissolved in saline.

A total of 12 cats were divided into three vaccine groups as follows:

-   -   Group 1 (naked DNA): Two injections, spaced 8 weeks apart, of        300 μg of naked hGH nucleic acid molecule in 500 μl saline.    -   Group 2 (LTI lipid 781-1): Two injections, spaced 8 weeks apart,        of 300 μg hGH nucleic acid molecule complexed with 150 μg        cationic lipid.    -   Group 3 (LTI lipid 106-3): Two injections, spaced 8 weeks apart,        of 300 μg hGH nucleic acid molecule complexed with 300 μg        cationic lipid.

At day 54 post injection, the cats were boosted with another injectionof the appropriate cationic lipid-DNA mixture. At day 111, cats wereboosted again, and at day 119, T-cell proliferation assays wereperformed as described. A T-cell stimulation index of 2 is taken as thecutoff and values below 2 are considered non-responsive.

Sera samples were collected from cats following the primary and boosteradministrations of complex as described in Example 3 and were assayedfor hGH specific antibody responses by ELISA. Endpoint ELISA titers areshown in Table 1. The lowest sera titers measured were 1:40. Therefore,negative titers are expressed as <1:40. TABLE 1 hGH antibody titers ofsera samples collected from cats administered a naked DNA vaccine or acomplex of the present invention encoding hGH Titer Titer T-cell at day54 at day 64 stimulation cat # Formulation (post prime) (post boost)index 1 Naked DNA <1:40 <1:40 9.1 2 Naked DNA <1:40 <1:40 2.4 3 NakedDNA <1:40  1:1125 12.7 4 Naked DNA <1:40 <1:40 11.3 geometric geometricmean = 40 mean = 92 5 DNA + lipid 781-1 <1:40 <1:40 1.6 6 DNA + lipid781-1  1:160  1:10,240 20.2 7 DNA + lipid 781-1  1:2312  1:7762 3.7 8DNA + lipid 781-1  1:233  1:21,183 8.9 geometric geometric mean = 242mean = 2865 9 DNA + lipid 106-3  1:1076  1:19,000 24.3 10 DNA + lipid106-3  1:316  1:19,135 65.5 11 DNA + lipid 106-3 <1:40 <1:40 18.2 12DNA + lipid 106-3  1:125  1:8693 2.6 geometric geometric mean = 203 mean= 3353

The results in Table 1 indicate that there was no seroconversion in anyof the four cats administered a single inoculation of 300 μg of thenaked hGH nucleic acid molecule. Moreover, following the boosteradministration, only one of the four cats in the naked DNA vaccine groupdeveloped an hGH-specific antibody response. In contrast to the nakedDNA vaccine group, 75% of the cats (i.e., 3 of 4 cats) cats in each ofthe two lipid groups developed detectable titers following the primaryadministration of complex, and these responses went up markedlyfollowing the booster administration of complex.

T-cell proliferation, measured by the T cell stimulation index,indicates that all treatments, including treatment with DNA alone,appeared to activate cell-mediated immunity. Treatment with a complex ofDNA and lipid 106-3 appears to work better for stimulating T cellproliferation in cats than did naked DNA alone or DNA complexed withlipid 781-1.

Example 6

This Example compares immune responses elicited using a nucleic acidmolecule encoding rabies glycoprotein G complexed with several cationiclipids of the present invention to the immune response elicited using anaked DNA vaccine encoding rabies glycoprotein G in cats.

This example compared the abilities of the following compositions toelicit an immune response against rabies glycoprotein G (rabies G) incats: a naked DNA vaccine consisting of the rabies G nucleic acidmolecule; and complexes between the rabies G nucleic acid molecule andone of the following cationic lipids: LTI lipid 106-3, LTI lipid 781-1,or LTI lipid 4518-52, each at a variety of DNA:lipid ratios. Also testedwas a complex that had been dehydrated by lyophilization and rehydratedprior to administration. Each of the compositions was produced asdescribed in Example 2. All cats received two intramuscular injectionsas described in Example 3, spaced four weeks apart. The following groupsof 4 cats each were tested:

-   -   Group 1: Naked DNA, 300 μg rabies G vector    -   Group 2: 300 μg lipid 781-1+300 μg rabies G vector    -   Group 3: 150 μg lipid 781-1+300 μg rabies G vector    -   Group 4: 75 μg lipid 781-1+300 μg rabies G vector    -   Group 5: 600 μg lipid 106-3+300 μg rabies G vector    -   Group 6: 300 μg lipid 106-3+300 μg rabies G vector    -   Group 7: 150 μg lipid 106-3+300 μg rabies G vector    -   Group 8: 300 μg lipid 4518-52+300 μg rabies G vector    -   Group 9: 300 μg lipid 106-3+300 μg rabies G vector (lyophilized        and rehydrated)    -   Group 10: 75 μg lipid 106-3+75 μg rabies G vector.

Group 1 served as a control group to demonstrate immunogenicity of thenaked DNA vaccine. Groups 2-4 were designed to determine if differencesin the lipid-to-DNA ratio were important for lipid 781-1. Similarly,groups 5-7 were designed to determine if differences in the lipid-to-DNAratio were important for lipid 106-3. Group 8 was included to examinethe efficacy of LTI lipid 4518-52. Group 9 was included to determine iflyophilization and rehydration of lipid:DNA complexes would improvecationic lipid vaccine efficacy in cats as previously demonstrated inmice by Gregoriadis, ibid. Finally, group 10 was included to determineis less than 300 μg of DNA could be used without affecting the abilityof lipid 106-3 to enhance the ability of cats to elicit an immuneresponse.

Rabies virus-specific neutralizing antibody activity was measured in thesera of all cats before and after the booster administration by RFFIT.Sera dilutions tested ranged from 1:5 to 1:174,693. Negative responsesare listed as a titer of <1:5 while responses that are stronger than thefinal dilution tested are indicated by the “>” sign. Injections weremade intramuscularly. It is known to those skilled in the art that ananti-rabies G antibody titer of 1:5 or greater, as measured by RFFIT, isprotective. Results from these studies are shown in Table 2. TABLE 2Rabies G antibody titers of sera samples collected from catsadministered a naked DNA vaccine or a complex of the present inventionencoding rabies G. Titer Titer cat # Formulation Pre-boost Post-boostGroup 1 QHR5 Naked DNA (300 μg rabies G) <1:5  1:25 BWM3 Naked DNA (300μg rabies G) <1:5 <1:5 3042 Naked DNA (300 μg rabies G) <1:5  1:1400ABO2 Naked DNA (300 μg rabies G) <1:5 <1:5 Group 2 QHH1 300 μg DNA + 300μg lipid 781-1  1:7000  1:167,449 3102 300 μg DNA + 300 μg lipid 781-1 1:1800  1:167,449 S72 300 μg DNA + 300 μg lipid 781-1 <1:5  1:50 QJB1300 μg DNA + 300 μg lipid 781-1 <1:5  1:230 Group 3 QHN4 300 μg DNA +150 μg lipid 781-1 <1:5  1:1400 QIN5 300 μg DNA + 150 μg lipid 781-1 1:2200  1:42,724 QGN5 300 μg DNA + 150 μg lipid 781-1  1:625  1:113,264QHG1 300 μg DNA + 150 μg lipid 781-1 <1:5  1:7000 Group 4 QHR1 300 μgDNA + 75 μg lipid 781-1 <1:5  1:50 QIN2 300 μg DNA + 75 μg lipid 781-1 1:280  1:5100 QGR5 300 μg DNA + 75 μg lipid 781-1  1:2400  1:6000 ACN1300 μg DNA + 75 μg lipid 781-1 <1:5  1:125 Group 5 3603 300 μg DNA + 600μg lipid 106-3  1:7000  1:174,693 BNJ2 300 μg DNA + 600 μg lipid 106-3<1:5  1:1800 ZAH1 300 μg DNA + 600 μg lipid 106-3  1:5100  1:159,751S203 300 μg DNA + 600 μg lipid 106-3  1:6300  1:67,491 Group 6 3525 300μg DNA + 300 μg lipid 106-3  1:280  1:45,668 BNJ1 300 μg DNA + 300 μglipid 106-3  1:125  1:6800 BMX2 300 μg DNA + 300 μg lipid 106-3 <1:5 1:6800 S197 300 μg DNA + 300 μg lipid 106-3 >1:167,449  1:6800 Group 73553 300 μg DNA + 150 μg lipid 106-3  1:1100  1:53,888 BNI3 300 μg DNA +150 μg lipid 106-3 <1:5  1:3125 E490 300 μg DNA + 150 μg lipid 106-3 1:25  1:6800 S192 300 μg DNA + 150 μg lipid 106-3  1:40  1:6000 Group 83541 300 μg DNA + 300 μg lipid  1:45  1:7000 4518-52 BNH4 300 μg DNA +300 μg lipid  1:1200  1:142,858 4518-52 BLR1 300 μg DNA + 300 μg lipid 1:280  1:7000 4518-52 S189 300 μg DNA + 300 μg lipid >1:7000  1:159,7514518-52 Group 9 DNA + lipid 106-3, dehyd  1.2700  1:142,858 &.rehyd¹BNF4 DNA + lipid 106-3, dehyd  1:170 >1:167,449 &.rehyd¹ E457 DNA +lipid 106-3,  1:1400  1:8,125 dehyd &.rehyd¹ S186 DNA + lipid 106-3,dehyd  1:45  1:3,125 &.rehyd¹ ¹300 μg rabies G DNA + 300 μg lipid106-3/dehydrated and rehydrated by the method of Gregoriadis, et al.,ibid. Group 10 BMC1 DNA, 75 μg + lipid 106-3, 75 μg  1:1800  16000 E451DNA, 75 μg + lipid 106-3, 75 μg  1:3125  1:38,206 QNV1 DNA, 75 μg +lipid 106-3, 75 μg  1:360  1:34,600 ZAF1 DNA, 75 μg + lipid 106-3, 75 μg 1:440  1:5,400 Geometric Mean Titers for each group, pre-boost and postboost, for each group mean mean titer, titer, Group Formulation pre-post- 1 Naked DNA (300 μg) <5 30.6 2 DNA, 300 μg + lipid 781-1, 300 μg(1:1) 133 4238 3 DNA, 300 μg + lipid 781-1, 150 μg (1:0.5) 77 14,756 4DNA, 300 μg + lipid 781-1, 75 μg (1:0.25) 64 661 5 DNA, 300 μg + lipid106-3, 600 μg (1:2) 1029 42,910 6 DNA, 300 μg + lipid 106-3, 300 μg(1:1) 413 10,409 7 DNA, 300 μg + lipid, 106-3, 150 μg (1:0.5) 48 9104 8DNA, 300 μg + lipid 4518-52, 300 μg (1:1) 570 32,518 9 DNA, 300 μg +lipid, 106-3, 412 49,159 300 μg dehyd& rehyd¹ 10  DNA, 75 μg + lipid106-3, 75 μg (1:1) 972 14,385

The data presented in Table 2 support the following conclusions: (1) inthe cats receiving the naked DNA vaccine, no seroconversion was observedfollowing the primary administration of vaccine immunization. Incontrast, all of the cats receiving a nucleic acid molecule-cationiclipid complex of the present invention exhibited seroconversion afterthe booster administration, and at least 50% of the cats seroconvertedper group after the initial administration of the complex. The bestseroconversion was seen in groups 8, 9, and 10 in which 100%seroconversion was observed following the primary administration ofcomplex. These results (0% seroconversion in group 1 and 100%seroconversion in groups 8-10 following the primary administration) werestatistically significant by Fisher's exact test (P<0.05). (2) Followingthe booster administration, all nine groups that were administered acomplex of the present invention exhibited stronger responses than thenaked DNA vaccine control group. Despite the small number of cats ineach group, statistically significant enhancement by Student's t testwas observed in groups 5 and 9 as compared to group 1, i.e. naked DNAvaccine. (3) Varying the ratio of lipid-to-DNA did not have significantimpact on the degree of enhancement (groups 2-4 and 5-7). (4)Dehydration and rehydration of the lipid:DNA complexes (lipid 106-3)prior to inoculation resulted in 100% seroconversion following theprimary administration and very strong responses in all cats followingthe boost (group 9). (5) Reducing the DNA dosage to 75 μg from 300 μgdid not result in any loss of the enhancement potential since 100%seroconversion was observed after the primary administration of thecomplex and very strong responses were observed in all cats post-boost(group 10).

Example 7 Measuring Luciferase Expression in Cat Muscle

Muscle and lymph node tissues were dissected and removed from the thighof a sacrificed cat, see Example 3. The tissues were quick frozen on dryice, and ground to a powder in liquid nitrogen. Ground frozen tissue wasresuspended in 1× cell culture lysate reagent (25 mM Tris-Phosphate, pH7.8, 2 mM DTT, 2 mM 1,2 diaminocyclohexane-N,N,N′,N′-tetraacetic acid,10% glycerol, 1% Triton X-100). After lysis, the cell debris was removedby centrifugation and supernatant was used in the following assay. Analiquot of the supernatant was mixed with Luciferase Assay Reagent,(Promega, Madison, Wis.). The mixture was placed in a Turner DesignsLuminometer TD-20/20, (Promega), and the light emitted was measured for15 seconds. The standard used to calibrate the assay was the recombinantfirefly luciferase QUANTILUM™, (Promega).

Example 8 Comparison of Expression of a DNA Plasmid, Formulated with andwithout LTI Lipid 106-3, in the Cat Muscle

In this example, evidence for increased antigen expression in the muscleupon formulation with lipid 106-3 was observed in an experiment in which300 μg of a plasmid vector encoding luciferase was injected into eachsemimembranosus muscle (inner thigh) of a cat, one muscle receiving DNAcomplexed with lipid, and one muscle receiving naked DNA. In the case ofDNA formulated with lipid 106-3, 300 μg of DNA was formulated with 300μg of lipid 106-3. Specifically, the right thigh of the cat was injectedwith DNA alone; the left thigh was injected with DNA formulated withlipid 106-3. After 48 hours, the cat was sacrificed, the muscles weredissected and luciferase activity was measured as described in Example7. Table 3 shows the luciferase assay standard curve used for thisexperiment, and Table 4 shows luminometer measurements for eachdissected tissue in the cat. TABLE 3 Luciferase assay standard curveSample Luminometer readings Blank 0.069 Positive control 332  2.5 μgstandard 387.9  250 nanogram(ng) standard 54.07   25 ng standard 9.817 2.5 ng standard 1.909  250 picogram (pg) standard 0.431   25 pgstandard 0.189

TABLE 4 Luminometer readings for each dissected muscle Amount of tissueused Luminometer muscle tested in luciferase assay reading Rightsuperficial muscle 140 milligram (mg) 0.044 (M. gracilis) Right deepmuscle 140 mg 0.065 (M. semimembranosus) Right Inguinal lymph node 100mg 0.040 Right Popliteal lymph node  73 mg 0.052 Left superficial muscle140 mg 0.058 Left Deep muscle 140 mg 13.82 Left Inguinal lymph nodecould not locate Not determined Left Popliteal lymph node  80 mg 0.078While no significant luciferase activity was observed in the leginjected with naked DNA, approximately 2 μg total of luciferase wasproduced in the entire deep muscle of the leg injected with thelipid/DNA formulation (assay sensitivity=2 pg), providing evidence forenhanced gene delivery and antigen production via use of cationic lipidformulations of the present invention.

Example 9 Effect of Cationic Lipid Formulated DNA Vaccines in Mice

This example demonstrates that formulation of DNA vaccines with cationiclipids does not enhance nucleic acid efficacy in mice, in contrast tothe enhancement of nucleic acid efficacy seen in cats treated withcationic lipid/DNA formulations.

Three different nucleic acid molecules, encoding rabies glycoprotein G,were prepared as described in Example 2. The first, pMV 5044, containsthe CMV intron A promoter, the rabies glycoprotein G coding sequence,and the rabbit beta globin polyadenylation sequence. The second, pMV5045, contains the CMV intron A promoter, the rabies glycoprotein Gcoding sequence, and the bovine growth hormone polyadenylation sequence.The third, pMV 5046, contains the CMV promoter, the rabies glycoproteinG coding sequence, and the bovine growth hormone polyadenylationsequence.

The three nucleic acid molecules encoding rabies glycoprotein G (rabiesG) were complexed with LTI lipid 106-3 at a lipid to DNA ratio (μg:μg)of 1:1 as described in Example 2. The corresponding “naked” DNA vaccineswere prepared by dissolving the plasmids in saline.

A total of 30 mice were divided into six vaccine groups as follows:

Group 1 (pMV 5044, 50 μg+lipid): One injection, intramuscular. Antibodytiters determined at four weeks post injection.

Group 2 (pMV 5044, 100 μg alone): One injection, intramuscular. Antibodytiters determined at four weeks post injection.

Group 3 (pMV 5045, 50 μg+lipid): One injection, intramuscular. Antibodytiters determined at four weeks post injection.

Group 4 (pMV 5045, 100 μg alone): One injection, intramuscular. Antibodytiters determined at four weeks post injection.

Group 5 (pMV 5046, 50 μg+lipid): One injection, intramuscular. Antibodytiters determined at four weeks post injection.

Group 6 (pMV 5046, 100 μg alone): One injection, intramuscular. Antibodytiters determined at four weeks post injection. TABLE 5 Anti-rabies Gantibody titers of sera samples collected from mice administered a nakedDNA vaccine or a complex of the present invention encoding rabies G.mouse # Formulation Titer Group 1 1 pMV5044, 50 μg + Lipid 106-3 1:40 2pMV5044, 50 μg + Lipid 106-3 1:40 3 pMV5044, 50 μg + Lipid 106-3 1:51 4pMV5044, 50 μg + Lipid 106-3 1:115 5 pMV5044, 50 μg + Lipid 106-3 1:135Group 2 1 pMV5044, 100 μg alone 1:43 2 pMV5044, 100 μg alone 1:53 3pMV5044, 100 μg alone 1:242 4 pMV5044, 100 μg alone 1:1060 5 pMV5044,100 μg alone 1:3795 Group 3 1 pMV5045, 50 μg + Lipid 106-3 1:40 2pMV5045, 50 μg + Lipid 106-3 1:65 3 pMV5045, 50 μg + Lipid 106-3 1:68 4pMV5045, 50 μg + Lipid 106-3 1:73 5 pMV5045, 50 μg + Lipid 106-3 1:137Group 4 1 pMV5045, 100 μg alone 1:1 2 pMV5045, 100 μg alone 1:46 3pMV5045, 100 μg alone 1:100 4 pMV5045, 100 μg alone 1:547 5 pMV5045, 100μg alone 1:640 Group 5 1 pMV5046, 50 μg + Lipid 106-3 1:1 2 pMV5046, 50μg + Lipid 106-3 1:1 3 pMV5046, 50 μg + Lipid 106-3 1:1 4 pMV5046, 50μg + Lipid 106-3 1:34 5 pMV5046, 50 μg + Lipid 106-3 1:54 Group 6 1pMV5046, 100 μg alone 1:1 2 pMV5046, 100 μg alone 1:1 3 pMV5046, 100 μgalone 1:1 4 pMV5046, 100 μg alone 1:1 5 pMV5046, 100 μg alone 1:59Rabies-virus specific neutralizing antibody activity was measured byRFFIT in the sera of all mice four weeks after injection with threedifferent nucleic acid molecules containing Rabies glycoprotein G.

The data presented in Table 5 indicate that cationic lipid formulationof a DNA vaccine does not enhance vaccine efficacy, as measured byhumoral (antibody) response, in mice. These data are in contrast toresults obtained in cats, where vaccine efficacy is enhanced byformulation with cationic lipids. For the nucleic acid constructpMV5044, formulation with lipid actually appears to slightly reduce DNAvaccine efficacy for mice, with the geometric means (of the five miceper group) declining from 294 with DNA alone to 66 with DNA/lipidcomplex. Results from the other two constructs in mice also showed noincrease in efficacy; the geometric means were as follows: for pMV5045,69.4 for DNA alone and 70.7 with DNA/lipid complex; and for pMV5046, 2.3for DNA alone and 4.5 for DNA/lipid complex.

Example 10 Administration of a DNA Plasmid, Formulated with and withoutLTI Lipid 106-3, to Cats

This example demonstrates the local immune response at the site ofinjection of DNA plasmids formulated with or without LTI lipid 106-3.Each of four cats was administered each of the following formulations toeach of the following sites on the ventral side: (a) saline (i.e.,vehicle alone) to the right arm; (b) 300 μg of lipid 106-3 (lipid alone)to the left arm; (c) 300 μg of a naked plasmid vector encoding rabiesglycoprotein G (naked rabies G vector) to the upper right foot; (d) 300μg of a naked plasmid vector encoding luciferase (naked luciferasevector) to the lower right foot; (e) 300 μg of rabies G vectorformulated with 300 μg of lipid 106-3 to the upper left foot; and (f)300 μg of luciferase vector formulated with 300 μg of lipid 106-3 to thelower left foot.

Six days after administration of the various formulations, the cats wereeuthanized and muscle and popliteal lymph node muscles were collected.Although the injection sites were marked, it was difficult to obtainmuscle samples from the injection sites; thus, only four injection siteswere identified, namely those for the saline only and naked rabies Gvector in one cat and those for lipid only and rabies G vector pluslipid in another cat. Muscle samples were sectioned using a cryostat andthe sections were stained using hematoxylin and eosin to analyze thepopulation of cells infiltrating the sites of injection. Muscle sampleswere also stained with antibodies specific for B-cells (anti-CD79aantibodies) using techniques known to those skilled in the art.

No differences were seen among the various lymph nodes with respect tocell infiltration. In the muscle samples where vehicle alone, lipidalone or naked rabies G vector was injected, the infiltrating populationof cells were mostly macrophage-like cells. In contrast, in the musclesample where the formulation comprising rabies G vector complexed withlipid was infected, the infiltrating cells were predominantlylymphocyte-like cells. Staining results with anti-CD79a antibodiessuggested that the majority of lymphocytes present were T cells.

These results, as well as others provided herein, suggest thatadministration of nucleic acid molecules complexed with cationic lipidsto cats leads to enhanced expression of the protein encoded by thenucleic acid molecule and infiltration of lymphocytes to the injectionsite which apparently does not occur when naked nucleic acid moleculesare administered in a similar manner. Without being bound by theory, itis believed that this infiltration of lymphocytes might explain theenhanced immune response seen with nucleic acid molecule-cationic lipidcomplexes of the present invention.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

1-27. (Canceled)
 28. A method to protect a felid from a disease, saidmethod comprising administering to said felid a composition comprising anucleic acid molecule complexed with a cationic lipid, wherein saidnucleic acid molecule encodes a protective compound.
 29. The method ofclaim 28, wherein said protective compound is an antigen.
 30. The methodof claim 28, wherein said protective compound is an antigen selectedfrom the group consisting of a protozoan antigen, a helminth parasiteantigen, an ectoparasite antigen, a fungal antigen, a bacterial antigenand a viral antigen.
 31. The method of claim 28, wherein said protectivecompound is an antigen selected from the group consisting of acalicivirus antigen, a coronavirus antigen, a herpesvirus antigen, animmunodeficiency virus antigen, an infectious peritonitis virus antigen,a leukemia virus antigen, a parvovirus antigen, a rabies virus antigen,a Bartonella antigen, a Yersinia antigen, a Dirofilaria antigen, aToxoplasma antigen, a flea antigen, a flea allergen, a midge antigen, amidge allergen, a mite antigen, a mite allergen and a tumor antigen. 32.The method of claim 28, wherein said cationic lipid comprisestetramethyltetraalkyl spermine analog lipid.
 33. The method of claim 28,wherein said felid is selected from the group consisting of domesticcats, lions, tigers, leopards, panthers, cougars, bobcats, lynx,jaguars, cheetahs, and servals.
 34. The method of claim 28, wherein saidfelid is a domestic cat.
 35. The method of claim 28, wherein said stepof administering said composition enhances an immune response relativeto the immune response generated by administration of a naked DNAvaccine encoding said protective compound.
 36. The method of claim 28,wherein said nucleic acid molecule:lipid ratio is from about 1:10 toabout 10:1.
 37. The method of claim 28, wherein said composition isadministered parenterally.
 38. A method to deliver a nucleic acidmolecule to a felid, said method comprising administering to said felida composition comprising a nucleic acid molecule complexed with acationic lipid, wherein said nucleic acid molecule encodes a protectivecompound.
 39. The method of claim 38, wherein said protective compoundis an antigen.
 40. The method of claim 38, wherein said protectivecompound is an antigen selected from the group consisting of acalicivirus antigen, a coronavirus antigen, a herpesvirus antigen, animmunodeficiency virus antigen, an infectious peritonitis virus antigen,a leukemia virus antigen, a parvovirus antigen, a rabies virus antigen,a Bartonella antigen, a Yersinia antigen, a Dirofilaria antigen, aToxoplasma antigen, a flea antigen, a flea allergen, a midge antigen, amidge allergen, a mite antigen, a mite allergen and a tumor antigen. 41.The method of claim 38, wherein said cationic lipid comprisestetramethyltetraalkyl spermine analog lipid.
 42. The method of claim 38,wherein said felid is selected from the group consisting of domesticcats, lions, tigers, leopards, panthers, cougars, bobcats, lynx,jaguars, cheetahs, and servals.
 43. The method of claim 38, wherein saidnucleic acid molecule:lipid ratio is from about 1:10 to about 10:1. 44.The method of claim 38, wherein said composition is administeredparenterally.
 45. A method to vaccinate a felid against an infectiousdisease, said method comprising administering to said felid acomposition comprising a nucleic acid molecule complexed with a cationiclipid, wherein said nucleic acid molecule encodes antigen selected fromthe group consisting of a calicivirus antigen, a coronavirus antigen, aherpesvirus antigen, an immunodeficiency virus antigen, an infectiousperitonitis virus antigen, a leukemia virus antigen, a parvovirusantigen, a rabies virus antigen, a Bartonella antigen, a Yersiniaantigen, a Dirofilaria antigen and a Toxoplasma antigen.
 46. The methodof claim 45, wherein said cationic lipid comprises tetramethyltetraalkylspermine analog lipid.
 47. The method of claim 45, wherein said step ofadministering said composition enhances an immune response relative tothe immune response generated by administration of a naked DNA vaccineencoding said protective compound.